Although a beating heart is vital for life, the factors which regulate contraction of cardiac muscle at the molecular level are not well understood. Recent evidence has shown that the caridac thick filament accessory protein myosin binding protein C (cMyBP-C) may have a regulatory role as well as a structural role in the thick filament. The importance of understanding the function of this protein is underscored by the fact that 25% of all patients with familial hypertrophic cardiomyopathy, an inherited cardiac disease, have a mutation in cMyBP-C. The goal of the proposed studies is to determine the importance of cMyBP-C for stabilizing the helically ordered arrangement of the myosin heads on the thick filament and for the binding of the giant elastic protein titin to the thick filament. We have developed a procedure for isolating cardiac muscle thick filaments that preserves the ordered arrangement of the myosin heads and structure. Recently, a knockout mouse for cMyBP-C in which cMyBP-C is absent from the muscle has been developed at the University of Wisconsin. This mouse provides a powerful system for examining the role of cMyBP-C in the thick filament and its relation to other proteins such as titin. The specific Aims of the project are to: 1 .) use the knockout mouse for cMyBP-C to study the effect of loss of cMyBP-C on the detailed structure of the isolated cardiac thick filament; 2.) use immunolabeling for titin to determine if it remains attached to the isolated cardiac thick filaments and to determine if cMyBP-C is necessary for its association with the thick filament; and 3.) use the recently developed technique of cryo-negative staining and single particle reconstruction to compute 3D-reconstructions of isolated mouse and rabbit cardiac thick filaments with sufficient resolution to show the precise arrangement of cMyBP-C relative to the myosin heads. Comparison of the reconstructions of the filaments from the normal mouse and the cMyBP-C knockout mouse will be used to definitively identify the location of cMyBP-C in the reconstructions. These studies will allow testing of the hypotheses that cMyBP-C is necessary to stabilize the helical arrangement of the crossbridge array and for binding of titin to the thick filament. The studies will also allow testing of the hypothesis that the location of cMyBP-C relative to the myosin head is consistent with its binding to the S2 region of the myosin head thus regulating contraction. These studies will provide significant information on the role of cMyBP-C in both the normal heart and in the diseased heart.